RESUMEN
Healthy cartilage maintenance relies on an equilibrium among the anabolic and catabolic processes in chondrocytes. With the onset of osteoarthritis (OA), increased interleukin (IL)-1ß levels induce an inhibition of the synthesis of extracellular matrix (ECM) proteins, as well as an increase in proteases. This eventually leads to a predominance of the catabolic phenotype and the progressive loss of articular cartilage. Hydrogen sulfide (H2S) is a small gaseous molecule recognized as the third endogenous gasotransmitter. When administered exogenously, it has shown anti-inflammatory and anti-catabolic properties in several in vitro and in vivo models. Here, OA cartilage disks were co-cultured in vitro with IL-1ß (5 ng/ml) and NaSH or GYY4137 (200 or 1000 µM) for 21 days. The ability of these two H2S-producing compounds to avoid long term extracellular matrix (ECM) destruction was evaluated. We used a glycosaminoglycan (GAG) quantification kit histology and immunohistochemistry (IHC) to evaluate matrix proteins degradation and matrix metalloproteinases (MMP) abundance. Through the GAGs quantification assay, safranin O (S-O) and toluidine blue (TB) stains, and keratan/chondroitin sulfate (KS/ChS) IHCs it was shown that co-stimulation with H2S-forming reagents effectively avoided GAGs destruction. Both Masson's trichrome (MT) stain and collagen (col) type II IHC, as well as aggrecan (agg) IHC demonstrated that not only were these proteins protected but even promoted, their abundance being higher than in the basal condition. Further, stains also demonstrated that positivity in the inter-territorial and intra-cellular for the different matrix components were rescued, suggesting that NaSH and GYY4137 might also have pro-anabolic effects. In addition, a clear protective effect against the increased MMPs levels was seen, since increased MMP3 and 13 levels were subsequently reduced with the co-stimulation with sulfide compounds. In general, GYY4137 was more effective than NaSH, and increasing the dose improved the results. This study demonstrates that H2S anti-catabolic effects, which had been previously proven in short-term (24-48 h) in vitro cellular models, are maintained over time directly in OA cartilage tissue.
Asunto(s)
Cartílago Articular/efectos de los fármacos , Cartílago Articular/metabolismo , Sulfuro de Hidrógeno/farmacología , Osteoartritis/tratamiento farmacológico , Osteoartritis/metabolismo , Sustancias Protectoras/farmacología , Cartílago Articular/patología , Glicosaminoglicanos/análisis , Glicosaminoglicanos/metabolismo , Humanos , Inmunohistoquímica , Interleucina-1beta/antagonistas & inhibidores , Interleucina-1beta/farmacología , Proteínas Matrilinas/análisis , Proteínas Matrilinas/metabolismo , Metaloproteinasa 13 de la Matriz/análisis , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/análisis , Metaloproteinasa 3 de la Matriz/metabolismo , Morfolinas/farmacología , Compuestos Organotiofosforados/farmacología , Osteoartritis/patología , Sulfuros/farmacología , Factores de TiempoRESUMEN
OBJECTIVE: Hydrogen sulfide (H2S), the third gasotransmitter together with NO and CO, is emerging as a regulator of inflammation. To test if it might offer therapeutic value in the treatment of osteoarthritis (OA) we evaluated the effects of two exogenous sources of H2S, NaSH and GYY4137, on inflammation and catabolic markers that characterize OA. METHOD: Human chondrocytes (CHs) were isolated from OA tissue. Cells were stimulated with a pro-inflammatory cytokine (interleukin-1ß, IL1ß, 5 ng/ml) and the ability of the two H2S sources to ameliorate its effects on the cells was tested. Nitric oxide (NO) production was quantified through the Griess reaction. Protein levels of inducible NO synthase (NOS2) and matrix metalloproteinase 13 (MMP13) were visualized through immunocytochemistry (ICC). Relative mRNA expression was quantified with qRT-PCR. Prostaglandin-2 (PGE-2), interleukin 6 (IL6) and MMP13 levels were measured with specific EIAs. NFκB nuclear translocation was visualized with immunofluorescence. RESULTS: Both H2S sources led to significant reductions in NO, PGE-2, IL6 and MMP13 released by the cells and at the protein level. This was achieved by downregulation of relevant genes involved in the synthesis routes of these molecules, namely NOS2, cyclooxigenase-2 (COX2), prostaglandin E synthase (PTGES), IL6 and MMP13. NFκB nuclear translocation was also reduced. CONCLUSION: NaSH and GYY4137 show anti-inflammatory and anti-catabolic properties when added to IL1ß activated osteoarthritic CHs. Supplementation with exogenous H2S sources can regulate the expression of relevant genes in OA pathogenesis and progression, counteracting IL1ß pro-inflammatory signals that lead to cartilage destruction in part by reducing NFκB activation.
Asunto(s)
Cartílago Articular/patología , Condrocitos/patología , Sulfuro de Hidrógeno/metabolismo , Inflamación/patología , Interleucina-1beta/farmacología , Metabolismo/efectos de los fármacos , Osteoartritis/patología , Sulfuros/farmacología , Anciano , Anciano de 80 o más Años , Biomarcadores/metabolismo , Cartílago Articular/efectos de los fármacos , Cartílago Articular/metabolismo , Células Cultivadas , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Dinoprostona/metabolismo , Femenino , Humanos , Inflamación/metabolismo , Interleucina-6/metabolismo , Masculino , Metaloproteinasa 13 de la Matriz/metabolismo , Morfolinas/farmacología , FN-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Compuestos Organotiofosforados/farmacología , Osteoartritis/metabolismoRESUMEN
The development of a calcium phosphate cement, comprising tetracalcium phosphate (TTCP) and dicalcium phosphate dihydrate (DCPD), that hardens in 14 min with water as the liquid or 6 min with a 0.25 mol/L sodium phosphate solution as the liquid, without using hydroxyapatite (HA) seeds as setting accelerator, is reported. It was postulated that reduction in porosity would increase cement strength. Thus, the effects of applied pressure during the initial stages of the cement setting reaction on cement strength and porosity were studied. The cement powder comprised an equimolar mixture of TTCP and DCPD (median particle sizes 17 and 1.7 microm, respectively). Compressive strengths (CS) of samples prepared with distilled water were 47.6 +/- 2.4 MPa, 50.7 +/- 4.2 MPa, and 52.9 +/- 4.7 MPa at applied pressures of 5 MPa, 15 MPa, and 25 MPa, respectively. When phosphate solution was used, the CS values obtained were 41.5 +/- 2.3 MPa, 37.9 +/- 1.7 MPa, and 38.1 +/- 2.3 MPa at the same pressure levels. Statistical analysis of the results showed that pressure produced an improvement in CS when water was used as liquid but not when the phosphate solution was used. Compared to previously reported TTCP-DCPD cements, the greater CS values and shorter setting times together with a simplified formulation should make the present TTCP-DCPD cement a useful material as a bone substitute for clinical applications.